Steroid modification with immobilized mycelium of Aspergillus phoenicis

Summary The bioconversion of progesterone by Aspergillus phoenicis has been studied. The metabolism in our conditions of experiment gave five important products. About 10 hours were necessary to obtain the highest concentration of 11--hydroxyprogesterone. During this time, 90% of progesterone were transformed at the optimal pH value 2.5. The transformations were tested using different immobilization methods which merits and limitations will be discussed.     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Production and localization of cellulases and β-glucosidase from the thermophilic fungusThielavia terrestris

Summary The enzyme production and localization ofThielavia terrestris strains C464 and NRRL 8126 were compared to determine their optimum temperature and pH for cellulase activity. High levels of intracellular -glucosidase activity were detected in the former strain. The intracellular -glucosidase of both strains were more thermostable than the extracellular enzyme; the half life ofT.terrestris (C464) endoglucanase activity at 60°C was greater than 96 hrs.     

Thermostable tryptophan synthase ofBacillus stearothermophilus expressed inEscherichia coli

Summary The tryptophan synthase genes,trpA andtrpB, from a moderate thermophile,Bacillus stearothermophilus IFO13737, were expressed efficiently inEscherichia coli. The recombinant tryptophan synthase amounted to 22% of the soluble cellular protein, and was purified to homogeneity by three steps. The enzyme is more thermostable thanE.coli tryptophan synthase, especially the subunit. The enzyme is also more resistant to sodium dodecylsulfate and methanol thanE.coli enzyme.     

Resistance of bacterial film to contamination during use as inoculum in repeated batch fermentation

Summary AnEscherichia coli strain constitutive for -galactosidase was immobilized onto cotton cloth. The resultingE.coli film was used as a resident inoculum in repeated batch fermentations for 30 days in the presence ofBrevibacterium ammoniagenes added as a contaminant. Analysis of -galactosidase production shows that contamination did not decrease the capacity of the film to generateE.coli cells, or decrease theE.coli population on the film.     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

Vertical Rotating Immobilized Cell Reactor of the bacteriumZymomonas mobilis for stable long-term continuous ethanol production

Summary Vertical Rotating Immobilized Cell Reactor was designed and built for glucose conversion into ethanol. Immobilized biomass units withZ. mobilis cells attached into polyurethane foam discs were fixed along a rotating shaft inside the bioreactor. The effect of rotation speed on the concentration of immobilized biomass was studied. Stability of the bioreactor over long-term operation was dependent on the concentration of the immobilized biomass. With fermentation carried out at 6 rpm a constant active immobilized cell concentration of only 34.5 g/l was maintained and used to convert up to 140 g glucose/l into more than 70 g ethanol/l with a volumetric ethanol productivity of 63 g/l/h.     

Recovery of plants from leaf protoplasts of hybrid-poplar and aspen clones

Leaf protoplasts were isolated from shoot cultures of two hybrid poplar clones (Populus alba × P. grandidentata Crandon, NC-5339 and P. nigra Betulifolia × P. trichocarpa, NC-5331) and the Upright European Aspen (P. tremula Erecta) and were cultured in contact with screen discs floated in liquid medium. Protoplast culture was influenced by the growth medium of the source shoot cultures, the protoplast purification procedure, the plating density, and the presence or absence of a coconut water and casein hydrolysate supplement added to the culture medium. The protoplast-derived cells divided more quickly and with higher incidence than previously reported for hybrid poplars. Shoots were regenerated from the protoplast-derived calli and were maintained as shoot cultures. Plants were developed from microcuttings rooted ex vitro and were grown-on in the greenhouse and field.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - NC-XXXX North Central Forest Experiment Station accession number assigned to hybrid poplar clones.     

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Isolation of aClostridium thermocellum gene encoding a thermostable β-1, 3-glucanase (laminarinase)

Summary AClostridium thermocellum gene directing the synthesis of a thermostable -glucanase was localized on a 1.9-kb DNA fragment by subcloning intoEscherichia coli plasmid vectors. The enzyme was highly efficient in degrading glucans with alternating -1, 3- and -1,4-linkages such as lichenan and barley glucan. It was also active towards the -1, 3-glucan laminarin, but lacked activity on cellulosic substrates and -glucans. The enzyme was therefore classified as -1, 3-glucanase (laminarinase) and the corresponding gene was designatedlicA. With barley -glucan as substrate the enzyme had a pH optimum around pH 6.5 and a temperature optimum at 65°C. It was stable for several hours at 60°C in the absence of substrate.     

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.     

Production of active human interferon-β inE. coli II. Optimal condition for induced synthesis by 3,β-indoleacrylic acid

Summary The maximum level of human interferon- activity was expressed under the control of theE. coli tryptophan promoter whenE. coli cells were induced at late logarithmic growth phase by 3,-indoleacrylic acid (IAA). The level is one order of magnitude higher than that obtained when the cells were induced at early logarithmic or stationary phase. When IAA was subsequently further added, the decrease in the activity observed at a latter period of fermentation was suppressed.     

Effects of “skeleton” photoperiods and high frequency light-dark cycles on the rhythm of cell division in synchronized cultures of Euglena

Summary Two further lines of evidence support the contention (Edmunds, 1966) that the cell cycle in autotrophically grown Euglena can be coupled to an endogenous, circadian biological clock under certain conditions. So-called skeleton photoperiods (LD: 3,6,3:12 and LD: 4,4,4:12) following a complete photoperiod regime entrain the cell division rhythm in the population to a precise 24 hr period, although the step-sizes of the successive fission bursts are always less than 2.00, indicating that not all cells divide in any one 24 hr interval. These findings imply that the continuous action of light is not required for synchronization and suggest that the putative oscillation underlying the rhythm can be phased by discrete light (or dark) pulses or signals.The effects of high frequency LD cycles whose periods were integral submultiples of 24 hr were also investigated. In most regimes (LD:1/4,1/2; LD:1/2,1; LD: 1,2; LD: 1,3; LD: 2,4; LD: 2,6; LD: 4,4) synchronous cell division iccurred in the culture with an average period of 26–27 hr, although only a fraction of the cells divided during any one burst. Similar results were obtained if (i) a synchronized culture was exposed to certain high frequency cycles whose periods were not integral submultiples of 24 hr (e.g., LD: 5,5 or LD: 8,8); (ii) an asynchronous culture (grown in LL) was subsequently exposed to a high frequency cycle; or (iii) a synchronized culture was subjected to a random LD cycle. The synchrony does not break down as long as the given LD regime is imposed and shows some indications of persistence in certain ensuing conditions of continuous illumination.A general formula was derived which predicts the time of division, t _D , for an individual cell: t _D =k+n, where k is the initial phase delay, n is an integer, and is the free-running period of the rhythm observed in the population. These results are interpreted as indicating that the high frequency cycles employed were unable to entrain the circadian oscillation(s) hypothesized to underly and gate cell division, with the result that the rhythm reverted to its free-running period. Exposure to such cycles, however, apparently either initiates a rhythm or synchronizes the phases of the individual oscillations in the populations of cells. The possible direct interaction between energy supply and the observed somewhat variable period lengths is discussed; also, the relevance of stochastic models for the decay of division synchrony in the absence of a recurrent synchronizing procedure is considered.Some of these results were initially reported at the 5th International Congress on Photobiology, Hanover, N.H., U.S.A., August 26–31, 1968.This work was supported by NSF research grants #GB-4140 and #GB-6892 to L. Edmunds.     

Spectrophotometric titrations of ferricytochrome C with ferrohexacyanide in the pH range 5 to 7

Spectrophotometric titrations were conducted on the system horse heart ferricytochromec plus ferrohexacyanide in the pH range 5 to 7 and at temperatures 8, 18, 22 and 28°C. A difference extinction coefficient for reducedvs. oxidized cytochromec at 550 nm of 21 mmol^–1cm^–1 was used in part of the evaluations. On the assumption that only one electron-transferlinked proton dissociation is effective for both ferro- and ferricytochromec in this pH range, various possible models are developed with only three conforming with the experimental pH dependence of the spectrophotometric equilibrium constant. The data conform best to a model with protonic dissociation constants between pH 5 and 7 such that the reduced cytochromec species is at least a factor of 3 more acidic than the one for oxidized cytochromec (with pK_H 6). This interpretation holds least for the data at 22°C, which points to a structural rearrangement at about this temperature (Czerlinski and Bracokova, 1973; Zabinski and Czerlinski, 1974; Zabinski, et al., 1974). While the extinction coefficient of ferrocytochromec shows no significant change with pH and temperature, the one for ferricytochromec does: it is about 5% larger at pH 5 than at pH 7 (550 nm). Graphs for the absorption change of ferricytochromec (pH 7 as reference) document the details over the wavelength range 500 to 750 nm.     

Some factors influencing adoption of sheltered dogs

Abstract

In Italy the law forbids euthanizing shelter dogs unless they are severely ill or dangerous. This has created a problem: many dogs are housed for long periods of time in shelters. It is important to the welfare of these dogs for us to find methods to increase successful adoption rates. The aim of this study was to evaluate the effect of different management factors such as the number of dogs in a pen, Temporary Adoption Programs (TAPs), and animal-owner-related characteristics on successful adoptions of kenneled dogs. The study included 763 dogs, 92 of whom were dogs showing behavioral problems. The age of the dogs upon arrival at the shelter was the most important determinant for length of stay, with younger dogs being adopted faster (Kruskal-Wallis, H = 150.27; df = 3; n = 733; p < 0.001). Dogs up to six months of age (n = 73) were adopted more quickly than older dogs (average length of stay: 1.4 vs. 6.4 months). The year of admittance was also significant since dogs who were admitted in a year in which more dogs were brought to the shelter spent more time in the shelter before being re-homed (Kruskal-Wallis, H = 96.18; df = 2; n = 733; p < 0.001). Dogs' gender had no effect on length of stay (Mann-Whitney, U = 64563; Z = 0.81; p = ns; n¹ = 389; n² = 344). Temporary Adoption Programs had a significant positive effect in reducing the return rate when the final adopter was the same person who had “temporarily” adopted the dog (Fisher exact test, p = 0.0063). Return rate was also associated with behavioral problems. Fearful dogs were returned more often than dogs with other problems (Fisher exact test, p = 0.029). It is concluded that, although young age is the most important factor leading to quick adoption, programs which include increased human interaction, and special training for dogs with behavioral problems, could aid in the successful re-homing of shelter dogs.     

Acetone-butanol-isopropanol production byClostridium beijerinckii (synonym,Clostridium butylicum)

Isopropanol is a product of the organism formerly known as Clostridium butylicum, which is now included in the speciesClostridium beijerinckii. We tested 52 strains ofC.beijerinckii for their ability to produce acetone,n-butanol, and isopropanol. The 32 butanol-producing strains may be divided into two groups based on whether isopropanol was produced. Isopropanol-producing cultures accumulated acetone only transiently.     

Glucosylation of methyl β-D-arabinofuranoside with 6′-chloro-6′-deoxysucrose and immobilized Protaminobacter rubrum

Summary Transglucosylation byProtaminobacter rubrum using 6-chloro-6-deoxysucrose (1) and methyl -D-arabinofuranoside (2) as donor and acceptor, respectively, were examined. inhibition caused by 6-chloro-6-deoxy-D-fructose (4) was observed and could be greatly lightened in a borate buffer, where the yield of the disaccharide (3) increased by 1.35-fold.     

Protoplast fusion and the cell cycle

Protoplasts isolated from rapidly dividing cell suspension cultures of either Nicotiana sylvestris or tumor derived cultures of Crepis capillaris were fused by PEG or liposome treatments to form homokaryons. Analysis of binucleates by Feulgen microspectrophotometry, and autoradiography, has revealed that whereas fusion products of all cell cycle combinations occur, protoplasts of certain cycle phases participate in fusions more frequently than expected, and there is a slight predominance of like-with-like cycle combinations. It is argued that this tendency towards specificity of fusion may be explained by cycle related variation in surface charge on protoplasts, and the mechanisms of action of the fusogens used.Abbreviations PEG polyethylene glycol - CAPT tumorous cell culture of Crepis capillaris - NS-1 cell culture of Nicotiana sylvestris     

Astaxanthin formation by the yeastPhaffia rhodozyma on molasses

Summary Phaffia rhodozyma grown on 7–10% B or C garde molasses contained 2 to 3 times more astaxanthin than reported earlier for this red yeast. Yield of astaxanthin with 10% molasses as fermentation substrate was 15.3 g/ml which was about 3 times higher than with glucose and 2 times higher than with a sugar blend representative of the molasses.     

Ribosylation of 3-Methylguanine and the Relative Stability of its 7- and 9-α-D-Ribofuranosides

Abstract

Ribosylation of 3-methylguanine la was investigated by enzymatic and chemical methods. Compound la did not act as a substrate for purine nucleoside phosphorylase. N-2-Protected 3-methylguanines 4 and 6 underwent exclusive N-7 glycosylation by fusion and chloromercury methods to give 5 and 7. Fully acetylated 7-α-D-ribofuranoside 5 was also obtained by thermal transglycosylation of the corresponding 9-α-D-ribofuranoside 9. The reverse isomerization 5 → 9 did not occur. The differences in the relative stability towards acidic hydrolysis between 7- and 9-(α-D-ribofuranosyl)-3-methylguanines are distinctly higher than those described so far for the other 7-9 isomeric nucleosides.